The role of ROS in the hematopoietic system has been a subject that has received little investigation due to the hypoxic environment inherent in the bone marrow niche. Furthermore, it is not known whether or not oxidative damage accumulations play a role in the functional decline of HSCs associated with aging. Measuring DNA damage and ROS levels using the Fragment Length Analysis by Repair Enzyme (FLARE) assay, I show here that there are indeed significantly detectable levels of 8-oxoguanine, a lesion associated with ROS, present in both young and old murine HSCs. In an attempt to attenuate the presence of these lesions, a four-week treatment with the thiol-based antioxidant N-Acetyl-L-Cysteine was administered orally to mice. Analysis revealed significant decreases in oxidative lesions in both the young and old HSC compartment. Additionally, it was demonstrated that the NAC treatment significantly reduced number of baseline DNA breaks in old, but not young, HSCs. Together these results suggest that DNA damage accumulation is a dynamic process that changes as cells age. Further understanding of the role of ROS will help elucidate the importance of this type of DNA damage on the declining functional potential associated with aging.

Phenolic antioxidants reduce the effect of oxidative stress within cells. They are found in a various fruits, vegetables and as food additives to reduce spoilage. Consumption of antioxidants by humans has been linked with increased lifespan and reduced incidence of cancer and cardiovascular disorders (Cabrera et al. 2006; Kuriyama 2008). In cultured mammalian cells however, some of these phenolic antioxidants have been reported to generate reactive oxygen species (ROS), leading to chromosomal breakage (Long et al. 2007; Long & Halliwell 2001). It is clear then, that amongst this group of compounds, in vitro toxicological study is not a reliable prediction of human hazard. It is for this reason that the work described in this thesis was undertaken: the principal aim was to gain a better understanding of the reasons underlying this contradiction. It has been suggested that excessive ROS generated in vitro might be a result of the higher levels of oxygen (~20%) compared to (1-7%) in vivo: (Yusa et al. 1984; Turrens et al. 1982). With clearer understanding, new experimental approaches might be taken to highlight or reduce positive in vitro genotoxicity test results that might be considered misleading. A diverse set of test compounds was first chosen. It included polyphenolic (PPA), monophenolic (MPA) and non-phenolic antioxidants (NPA), in addition to mechanistically characterised oxidants, genotoxins and cytotoxic, non-genotoxins as controls. Genotoxicity was assessed in vitro using the GADD45a, GFP reporter assay and in silico using Derek Nexus™. Amongst the 19 antioxidants assessed, the 11 of 12 of PPAs, 0 of 4 MPAs and 1 of 3 NPAs (ethoxyquin) produced positive results in vitro and 8 of 12 PPAs generated alerts of at least plausible genotoxicity in silico. To discover whether these results were the result of cellular hyperoxia-promoted generation of physiologically irrelevant ROS in cells, genotoxicity was reassessed in the presence of 1 and 5% oxygen. This reduced oxygen exposure had no effect upon the qualitative result for any of the assessed compounds and a negligible effect upon the dose at which any positive result was produced. An assessment of the ability of antioxidants to generate potentially genotoxic ROS within cells was carried out using the intracellular fluorescent dye, dichlorofluorescin diacetate (DCFH-DA). 10 of 12 PPAs, 0 of 4 MPAs and 1 of 3 NPAs (ethoxyquin) were shown to increase the level of ROS within TK6 human lymphoblastoid cells within 4 hours of compound exposure. Within this same timeframe, the mitochondrial membranes in cells treated with 10 of 12 PPAs, 2 of 4 MPAs and 1 of 3 NPAs (ethoxyquin) were shown to become depolarised using JC-1 dye. It was unclear however, whether mitochondrial membrane depolarisation was a cause or a consequence of ROS generation within the cells. In order to assess whether the increase in intracellular ROS led to an increase in oxidised DNA within treated cells, 8-oxoguanine (8-OG) was quantified using a FITC conjugated anti8-OG antibody. This assessment revealed that levels of the oxidised base were only increased in cells exposed to two of the 12 PPAs (quercetin and resorcinol). The level of 8OG detected was lower than the vehicle control for cells treated with 10 of the 15 antioxidants. One interpretation of this is that these agents induce the repair pathway for oxidative damage, which leads to a lower level of oxidised DNA bases in the genome. The results showed that while a large proportion of PPAs produce genotoxic results in vitro and lead to increased levels of ROS, the amount of oxidised DNA is not higher in treated cells. This would suggest the presence of a different mechanism for the observed genotoxicity.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18546号 / 医博第3939号 / 新制||医||1006(附属図書館) / 31446 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武田 俊一, 教授 松田 文彦, 教授 小松 賢志 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

Studies described in this thesis were aimed at characterizing the mechanism(s) of aflatoxin B1 (AFB1) pulmonary carcinogenesis by addressing the formation, prevention, and repair of AFB1-induced oxidative DNA damage. The ability of AFB1 to cause oxidative DNA damage in different lung cell types of the A/J mouse was examined. The formation of 8-hydroxy-2’-deoxyguanosine (8-OHdG) in freshly isolated mouse lung alveolar macrophages, alveolar type II cells, and nonciliated bronchial epithelial (Clara) cells, was assessed by high performance liquid chromatography with electrochemical detection. An increase in 8-OHdG formation occurred in macrophage and Clara cell preparations isolated from A/J mice two hours following in vivo treatment with a single tumourigenic dose of AFB1. Prior treatment with polyethylene glycol-conjugated catalase (PEG-CAT) prevented the AFB1-induced increase in 8-OHdG levels in all mouse lung cell preparations. These results support the possibility that oxidative DNA damage in mouse lung cells contributes to AFB1 carcinogenicity. Mouse lung tumourigenesis was assessed following treatment of A/J mice with PEG-CAT and/or AFB1. Unexpectedly, the mean number of tumours per mouse and tumour size in the PEG-CAT + AFB1 group were greater than those of the group treated with AFB1 alone. There was no difference in K-ras exon 1 mutation spectrum or in the histological diagnosis of tumours between treatment groups. In vitro incubation with mouse liver catalase (CAT) resulted in conversion of [3H]AFB1 into a DNA-binding species, a possible explanation for the results observed in vivo. These results demonstrate that PEG-CAT is not protective against AFB1 carcinogenicity in mouse lung despite preventing DNA oxidation. The effect of in vivo treatment of mice with AFB1 on pulmonary and hepatic base excision repair (BER) activities and levels of 8-oxoguanine DNA glycosylase (OGG1) was investigated. AFB1 treatment increased 8-OHdG levels and BER activity in mouse lung, but did not significantly affect either in liver. Levels of OGG1 immunoreactive protein were increased in both mouse lung and liver. These results indicate that oxidative DNA damage may be an important mechanism in the carcinogenicity of AFB1. However, BER activity is increased by AFB1 treatment, possibly representing a compensatory response to the production of oxidative DNA damage. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2008-12-12 10:00:44.81

Silver nanoparticles (AgNPs) containing consumer products have increasingly emerged in the market because of their potential antibacterial property, which might result in increased human exposure and environmental contamination. AgNPs are toxic to mammalian and other cells but the determinants of this toxicity remain to be fully characterised and the potential impact of DNA repair systems has been poorly explored. This study, therefore, examined to what extent the size and shape of synthesised AgNPs determined AgNP toxicity in DNA repair proficient and deficient (8-oxoguanine DNAglycosylase; WT and OGG1-/-, respectively) mouse embryonic fibroblasts (MEFs) as well as a well-known human cell line used in the toxicity testing, HepG2 cells. Citrate-stabilised spherical- and triangular-shaped AgNPs (S-AgNPs andT-AgNPs, respectively) were synthesised chemically from AgNO3 using combinations of NaBH4 and sodium citrate as a reducing and stabilising agent, respectively, and purified by dialysis. Three different sized S-AgNPs were prepared with diameters of 7.6 ± 1.2, 14.3 ± 4.2, and 52.5 ± 17.9 nm as measured using transmission electron microscope (TEM), and their zeta potentials were -36.1±2.7, -39.5±2.7 and -36.7±4.1 mV, respectively. T-AgNPs had an edge length and thickness of 71.4 ± 11.1 nm and 5.7 ± 0.8 nm, respectively. The size and zeta potential of the purified AgNPs were constant in distilled water for at least 6 months. The uptake of both S- and T-AgNPs by cells resulted in a time and dose-dependent increase in the number of cellular AgNPs and the amount of Ag+ released intracellularly. These increases were associated with a decrease in cell viability (as measured using the MTT assay) and cell survival (the clonogenic assay), and an induction in ROS generation (the DCF assay) and DNA damage(the alkaline Comet assay) for all three cell lines. AgNPs were observed in cells using TEM, suggesting the uptake of AgNPs via an endocytosis pathway. Results suggested that an increase in cellular AgNP level and intracellular released Ag+ content were associated with a time and dose-dependent toxicity. Interestingly, cellular AgNP level and intracellular released Ag+ content might play an important role in size-dependent AgNP toxicity, in which exposure to the smaller S-AgNP sizes (7nm and 14nm) resulted in higher levels of both cellular AgNPs and Ag+ released intracellularly, and then to increased toxicity when compared with the larger S-AgNP size (50nm). Moreover, different shaped AgNPs might induce toxicity by different mechanisms: ROS-mediated toxicity might be induced by both 70nm T-AgNPs and 50nm S-AgNPs and 70nm T-AgNPs might also induce cell membrane damage. AgNP-induced toxicity was different in different cell lines with HepG2 cells being more sensitive to AgNPs particularly using the clonogenic assay, and this toxicity was associated with higher DNA damage observed in HepG2 cells after 24 h. OGG1-/- MEFs were more sensitive to intracellular released Ag+, leading to higher ROS formation and DNA damage in OGG1-/- MEFs than that observed in WT MEFs. In summary, this study strongly suggests that AgNPs induce toxicity via a Trojan-horse type mechanism, and not only Ag+ released intracellularly but also cellular AgNPs take part in this toxicity, and will eventually result in the biological responses of the cells.

The products of oxidative damage to double-stranded (ds) DNA initiated by photolytically generated sulfate radical anions SO4•− were analyzed using reverse-phase (RP) high-performance liquid chromatography (HPLC). Relative efficiencies of two major pathways were compared: production of 8-oxoguanine (8oxoG) and hydrogen abstraction from the DNA 2-deoxyribose moiety (dR) at C1,′ C4,′ and C5′ positions. The formation of 8oxoG was found to account for 87% of all quantified lesions at low illumination doses. The concentration of 8oxoG quickly reaches a steady state at about one 8oxoG per 100 base pairs due to further oxidation of its products. It was found that another guanine oxidation product identified as 2-amino-5-(2′-alkylamino)-4H-imidazol-4-one (X) was released in significant quantities from its tentative precursor 2-amino-5-[(2′-deoxy-β-d-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz) upon treatment with primary amines in neutral solutions. The linear dose dependence of X release points to the formation of dIz directly from guanine and not through oxidation of 8oxoG. The damage to dR was found to account for about 13% of the total damage, with majority of lesions (33%) originating from the C4′ oxidation. The contribution of C1′ oxidation also turned out to be significant (17% of all dR damages) despite of the steric problems associated with the abstraction of the C1′-hydrogen. However, no evidence of base-to-sugar free valence transfer as a possible alternative to direct hydrogen abstraction at C1′ was found.

The development of quantum mechanics has historically allowed researchers to theoretically explore the fundamental physical properties of atoms and molecules. Although quantum mechanics has been around for almost a century, its use was largely limited by the computational complexity it demanded. In the past decade, computer technology has evolved to the point where it is possible to perform calculations on biologically relevant systems. This has allowed us to corroborate results obtained from experiment as well as predict and explain phenomena that experiment cannot. Unfortunately, the field as a whole has not progressed to the point where high level methods, such as Multi-Reference Configuration Interaction (MRCI), are applicable to large molecular systems. Thus, to effectively study these systems, compromises must be made. In this work, two different approaches are taken to study the photophysical properties of systems such as DNA. In the first approach, a model system is formulated and studied in lieu of the larger target system. The excited state dynamics of 8-oxoguanine (8-oG) and its anion are studied in order to assess the possibility of taking part in an electron transfer mechanism to repair a nearby cyclobutane pyrimidine dimer (CPD). It is found that barriers on the anion S1 excited state surface prohibits easy access to conical intersections with the ground state, causing the anion to have a much longer excited state lifetime than the neutral form. Although much insight can be gained by this method, it is not uncommon for crucial interactions to be lost through simplification. In this case, when 8-oG is placed in an adenine dinucleotide, the π stacking interaction allows it to form a long lived radical base pair, which may be fundamental to its role in CPD repair. Unfortunately, it is impossible to carry out the same excited state calculations for the 8-oG/adenine dinucleotide due to computational cost. For reasons such as these, we also implement and benchmark a new approach to carrying out high level configuration interaction calculations in which the MRCI is expanded in the basis of high multiplicity natural orbitals (HMNOs). Specifically, the HMNO approach is implemented by expanding the MRCI wavefunction in the basis of natural orbitals generated from a ground state high multiplicity Configuration Interaction Singles and Doubles (CISD) calculation. Excited state calculations both at and away from the Franck-Condon region were performed to benchmark the ability of the HMNO approach using CISD and MRCI to reproduce standard MRCI energies. The ability of the HMNOs to be truncated was also explored, yielding efficient truncation criteria and guidelines for choosing the best basis set. It is found that the MRCI/HMNO approach yields energies that are in excellent agreement with standard MRCI while only requiring a fraction of the computational effort, possibly allowing it to be applied to larger systems such as nucleotide dimers. / Chemistry

The studies described in this thesis investigated the relationship between DNA repair mechanisms, aflatoxin B1 (AFB1)-induced DNA damage and carcinogenesis. Mice deficient in 8-oxoguanine glycosylase (OGG1, the rate-limiting enzyme in repair of oxidized guanine), mice heterozygous for OGG1, and wild type mice, were exposed to a single tumourigenic dose (50 mg/kg) of AFB1. Neither ogg1 genotype nor AFB1 treatment affected levels of oxidized guanine in lung or liver 2 h post-treatment. ogg1 (-/-) mice had increased susceptibility to AFB1 toxicity, as reflected by increased mortality within one week of AFB1 exposure. AFB1 treatment did not significantly increase lung or liver tumourigenesis compared to DMSO controls. No difference was observed between ogg1 genotypes, although a non-significant trend towards AFB1-treated ogg1 (-/-) mice being more susceptible to tumourigenicity was apparent. Overall, deletion of ogg1 did not significantly affect AFB1-induced DNA damage or tumourigenicity, suggesting that oxidized guanine may not be a major contributor to AFB1-induced tumourigenesis. The effects of AFB1 on DNA repair were assessed in p53 (a protein implicated in regulation of DNA repair) wild type and heterozygous mice. p53 (+/+) mice treated with 0, 0.2 or 1.0 ppm AFB1 for 26 weeks had increased nucleotide excision repair (NER) activities in lung and liver compared to control, which may represent an adaptive response to AFB1-derived DNA adducts. In p53 (+/-) mice, the AFB1-induced increase in NER was significantly attenuated, suggesting that loss of one allele of p53 limits the ability of NER to up-regulate in response to AFB1-induced DNA damage. Twenty-six week exposure to AFB1 did not affect base excision repair (BER) in p53 (+/+) mouse lung or liver compared to control. BER was significantly decreased in livers from mice exposed to 1.0 ppm AFB1 compared to those exposed to 0.2 ppm AFB1, a result that was not due to liver cell death or to altered levels of OGG1 protein. In lungs and livers of p53 (+/-) mice, BER activity was unchanged by AFB1. As such, the difference in BER response between 0.2 ppm and 1.0 ppm AFB1 treatment seen in the p53 (+/+) mice appears to be p53 dependent. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-10-17 22:24:31.577

DNA double-strand breaks (DSB) with chemically modified end-termini are the most significant lesions resulting from radio/chemotherapeutic intervention of cancer and non homologous end-joining (NHEJ) factor Artemis nuclease has been implicated in the repair of such breaks. To examine whether the resolution of terminally blocked DNA DSBs is the biologically relevant function of Artemis, Artemis deficient fibroblasts were stably complemented with wild type or an endonuclease deficient D165N mutant Artemis. Physiological levels of wild type (WT) Artemis completely restored DSB repair proficiency and resistance to γ-radiation, bleomycin, and neocarzinostatin. Cells expressing the D165N mutants remained as chemo/radiosensitive and as repair deficient as parental cells, with persistent γ H2AX and 53BP1 foci that increased in size 6-18 hour post irradiation. These persistent foci co-localized with DNA double strand break repair factor Mre11 and also with promyelocytic leukemia protein (PML). Further, in vitro studies have revealed that DNA-PK dependent Artemis endonucleolytic activity may play a role in the repair of commonly found oxidative base damage; 8-oxoguanine (8-oxoG), a hallmark of complex DSBs. However, majority of DNA DSBs are repaired in an Artemis independent manner, and recently discovered, DNA end-specific nuclease, Metnase is a candidate enzyme for repair of such breaks. To study the role of Metnase in resolution of 3ʹ-blocked termini, several substrates mimicking such breaks were constructed. A 3ʹ-phosphoglycolate moiety on longer overhangs (4 and 6 bases) altered specificity and stimulated Metnase-mediated cleavage of the terminal 3 nucleotides. However, an 8-oxoG residue at the single-strand/double-strand border did not affect specificity or extent of cleavage. Metnase preferentially cleaved ssDNA-overhang of a partially duplex substrate, and the cleavage increased with increase in length of 3ʹ-overhangs. A D483A mutation in Metnase completely abrogated Metnase cleavage activity towards DNA ends. These results suggest that Metnase may resolve oxidatively damaged DNA ends to facilitate repair while Artemis is required for the resolution of more complex DNA DSBs that persist for longer times and are not amenable to repair by other NHEJ factors.

The amidohydrolase superfamily is a functionally diverse group of evolutionarily related proteins which utilize metal cofactors in the activation of a hydrolytic water molecule and in the stabilization of the resulting tetrahedral intermediate. Members of this superfamily have been described which use one or two divalent transition metals. These metal cofactors are located in either or both of two active-site metal binding centers which are labeled as the Ma and MB sites. The goal of this research was to elucidate the nature of the reactions catalyzed by Ma and MB mononuclear members of the amidohydrolase superfamily. This was approached through comprehensive mechanistic evaluations of two enzymes which utilized the different metal sites. Nacetyl- D-glucosamine-6-phosphate deacetylase from E. coli (NagA) and cytosine deaminase from E. coli (CDA) served as models for mononuclear amidohydrolase superfamily enzymes which have evolved to utilize a single B-metal and a single a-metal for hydrolysis, respectively. This research elucidated the different properties imparted by the distinct a and B active sites and the specific interactions utilized by the enzymes for substrate binding and catalysis. These studies led to the eventual proposal of detailed chemical mechanisms and the identification of rate determining steps. Knowledge of sequence-function relationships was applied toward the discovery of function for enzymes related to cytosine deaminase and guanine deaminase. The first group of enzymes investigated was proposed to catalyze the fourth step in riboflavin and coenzyme F420 biosynthesis in Achaea. Three putative deaminases; Mm0823 from Methanosarcina mazei, MmarC7_0625 from Methanococcus maripaludis C7 and Sso0398 from Sulfolobus solfataricus were cloned and expressed. These proteins proved to be intractably insoluble. A second set of enzymes, Pa0142 from Pseudomonas aeruginosa PA01 and SGX-9236e (with crystal structure PDB: 3HPA) were found to catalyze the novel deamination of 8-oxoguanine, a mutagenic product of DNA oxidation. 9236e was cloned from an unidentified environmental sample of the Sargasso Sea. The closest homolog (98% identical) is Bcep18194_A5267 from Burkholderia sp. 383. Additionally, it was discovered that the proteins SGX-9339a (with crystal structure PDB: 2PAJ) and SGX-9236b catalyzed the deamination of isoxanthopterin and pterin-6- carboxylate in a poorly characterized folate degradation pathway. These enzymes were also from unknown environmental samples of the Sargasso Sea. The closest homolog of 9339a (88% identical) is Bxe_A2016 from Burkholderia xenovorans LB400. The closest homolog of 9236b (95% identical) is Bphyt_7136 from Burkholderia phytofirmans PsJN.